Project Summary/ Abstract Periosteal bone growth and modeling take place in the periosteum at the outer surface of cortical bones. The perioseum, which covers the entire bone surface except the portion of bones that contains articular cartilage, is one of the most osteogenic tissues in the body and plays a critical role in cortical expansion during growth. Periosteal bone formation has different mechanisms from other areas of bone surface such as endosteum, secondary spongiosa and trabecular bone remodeling, etc. Despite its physiological significance, the periosteum is often overlooked when it comes to processes that occur in the underlying cortical bone. Periosteal biology remains little investigated and poorly understood. Structurally, the periosteum is composed of two layers; adjacent to the periosteal bone surface is a layer of loosely packed cells essential for growth and repair of the underlying bone. Further outward is a layer of densely packed periosteum derived stem cells (PDSCs) interspersed with lymphatics, blood vessels and nerve endings. The microenvironment of the outer layer serves as a niche to maintain the PDSCs whilst the inner layer provides an osteogenic microenvironment for periosteal bone formation. We have shown that there are no osteal macrophages and Trap+ mononuclear cells in CSF-1 op/op mice (CSF-1-/-), and interestingly, no obvious cortical bone formation. A single injection of rhM-CSF is sufficient for rescue of osteoclast recruitment and survival in CSF-1-/- mice. These results suggest that periosteal macrophages are essential in maintenance of periosteum microenvironment for periosteal bone formation. Moreover, we have shown that Trap+ mononuclear cells derived from wild WT mice secrete PDGF-BB to induce migration of mesenchymal stromal/stem cells. Knockout of PDGF-BB in the Trap+ cell lineage reduces periosteal angiogenesis and bone formation but PDSCs and matrix proteins are still present in the periosteum, suggesting periosteal macrophages play more important role in maintenance of periosteum homeostasis. In addition, we have found mechanical stress induces periosteal TRAP+ mononuclear cells to secrete PDGF-BB for angiogenesis and bone formation in our preliminary data. Thus, we hypothesize that periosteal macrophages maintain homeostasis of the periosteum and TRAP+ mononuclear cells recruit PDSCs from outward layer to the periosteal surface for angiogenesis and bone formation. In this proposal, we will first determine the role of periosteal macrophages in periosteum homeostasis. We will then determine the function of Trap+ mononuclear cells, specifically, the mechanisms by which PDSCs are recruited by Trap+ mononuclear cells for periosteal bone formation. Finally, we will validate the role of TRAP+ mononuclear cells in mechanical stress-induced periosteal bone formation.